Chromatography Forum

Please could someone suggest why the below outcome occurred?

An analyst in hplc chromatography injects a 5ul and 10 ul injection volume into a valve injector and loads the loop manually with a syringe. to save time he decides to use a 10ul loop for both injections. For the 5ul injection he partially fills the loop using a syringe and for the 10ul injection he overloads the loop by loading 15ul into the syringe. He believes that the 15ul will ensure the loop is completely filled and his precision will be better than the 5ul injection. Why did the precision checks show the contrary i.e. the 5ul was better.

Please help me explain why the 5ul was better than 15ul injection into a 10ul loop. Suggest how the analyst could approach this better so the most precise injection of 5 and 10ul samples into the HPLC system. Thanks!

You didn't mention specific equipment, but lets assume that you are using a standard Rheodyne injector with a 10 uL loop. In general, for all loop injectors there are two operating modes: full loop and partial loop.

Partial loop mode means you fill up only a portion of the loop volume (10 uL in this case). Because of laminar flow inside the tubing (the sample in the middle of the tube goes through faster than the sample along the walls), you should only fill up to 50% of the loop volume (5 uL in this case). If you put more into the loop, some of the sample may be lost in the waste line.

For full loop mode, you must overfill the loop to make sure that you have flushed all solvent out of the tube, especially the sample next to the walls of the tube. To do this you need to add about 4 - 5 loop volumes of sample! That means 40 - 50 uL in this case. A 15 uL sample is not enough.

Other factors for manual injection: leave the syringe in the injector while turning the valve, be consistent in how fast you load the loop, flush it with solvent after injection.

Maybe a picture will help. This is an outtake from our "HPLC Basics, Equipment, & Troubleshooting" course

Hi

Other things to consider

How different are the two sets of results
Is the larger volume injector overloading
The solvent the sample is in

There are several other possible areas causing the differing results that are not directly related to the injector

The wording of that question sounds exactly like something off an exam paper. I do hope it wasn't...

Imh, did you want your statement to be at the "HPLC" post?

LCbob, are you talking about a solvent incompatibiliy? I am trying to figure out how that would cause a wrong area.

Imh, did you want your statement to be at the "HPLC" post?

I can't answer for lmh, but I thought the first post of this thread was also an exam question, as obviously was the HPLC question.

Bruce Hamilton

Imh, did you want your statement to be at the "HPLC" post?

LCbob, are you talking about a solvent incompatibiliy? I am trying to figure out how that would cause a wrong area.

Was thinking if too stronger solvent was used the higher volume may cause poor peak area and hence possible repro problems